Dasypyrum Villosum Translocations Lines in the Same Wheat Genetic Background

agronomy

Article Genotypic and Phenotypic Characterization of Two Triticum aestivum L.—Dasypyrum villosum Translocations Lines in the Same Wheat Genetic Background

Baicui Wang 1,2, Xiaolan Ma 1,2, Xingguo Ye 1,2, Yilin Zhou 3, Youzhi Ma 1,2 and Zhishan Lin 1,2,*

1 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China; [email protected] (B.W.); [email protected] (X.M.); [email protected] (X.Y.); [email protected] (Y.M.) 2 National Key Facility of Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China 3 Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; [email protected] * Correspondence: [email protected]

Abstract: A wheat 660K chip was used to genotype two wheat-Dasypyrum villosum 6V#4S.6DL and 6V#2S.6AL translocation lines (A303 and B303) and their common wheat recurrent parent Wan7107. The results showed that these three lines have similar characteristics of base composition except for the translocation chromosomes. The alien translocation chromosomes have fewer homozygous

and more heterozygous genotypes with more invalid probes. Distributions of SNPs between the translocation lines and Wan7107 were mainly dense on the regions of 6AS or 6DS as expected, but

Citation: Wang, B.; Ma, X.; Ye, X.; unexpectedly also on near the telomere of 2BS, and some regions of other wheat chromosomes. Zhou, Y.; Ma, Y.; Lin, Z. Genotypic Meanwhile, the translocation lines A303 and B303 have 99.44% and 98.81% identical genotypes and Phenotypic Characterization of to Wan7107, respectively. Under the same genetic background, A303 and B303 showed different Two Triticum aestivum L.—Dasypyrum reactions to Blumeria graminis f. sp. tritici (Bgt) strains of powdery mildew. Both translocation lines villosum Translocations Lines in the have higher grain weight and plant height, and B303 has fewer spikelets compared to Wan7107. Same Wheat Genetic Background. These results provide us a new insight into the genomic variation between the backcross generation Agronomy 2021, 11, 399. https:// plant and the recurrent parent, which is valuable information for understanding the relationship doi.org/10.3390/agronomy11020399 between wheat and the 6VS chromosome of D. villosum as well as the application potential of the alien chromosome arms. Academic Editors: Ahmad M. Alqudah and Andreas Börner Keywords: 660k chip; wheat; Dasypyrum villosum; translocation; powdery mildew resistance; agronomic traits Received: 21 January 2021 Accepted: 20 February 2021 Published: 23 February 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most published maps and institutional afﬁl- important food crops in the world, providing approximately 20% of the calories consumed iations. by humans [1]. Wheat is the second largest grain crop in China. However, wheat produc- tion globally and in China is facing numerous challenges including limited arable land, growing populations, climate change [2], and biotic stress [3]. Yield is directly affected by extreme changes in wheat growing environments and natural disasters, among which

Copyright: © 2021 by the authors. drought [4], heat [5], and diseases [6] are the most signiﬁcant. In order to broaden the Licensee MDPI, Basel, Switzerland. genetic basis of wheat to meet these challenges, mining and utilizing desired genes derived This article is an open access article from wild and related species for wheat improvement is now common. Genomes or chro- distributed under the terms and mosomes from related species such as Secale cereale L. [7–9], Dasypyrum villosum [10–15], conditions of the Creative Commons Aegilops species [16–19], Hordeum vulgare L. [20], and Thinopyron elongatum [21], which Attribution (CC BY) license (https:// contain a large number of wheat disease resistance genes, are tolerant to stress, have creativecommons.org/licenses/by/ good ﬂour-processing quality, and have been previously transferred into wheat to cre- 4.0/).

Agronomy 2021, 11, 399. https://doi.org/10.3390/agronomy11020399 https://www.mdpi.com/journal/agronomy Agronomy 2021, 11, 399 2 of 19

ate amphidiploids [22,23], addition lines [20,24], substitution lines [25], or translocation lines [10,26–28]. Powdery mildew, caused by the biotrophic parasitic fungus Blumeria graminis f. sp. tritici (Bgt), is an important wheat disease worldwide and can cause severe yield losses ranging from 5 to 40% [29,30]. Currently, 89 wheat powdery mildew resistance genes/alleles (Pm1- Pm65) have been cataloged, which are located on nearly all wheat chromosomes [31,32]. Forty-four of these genes originated from progenitors and wild relatives of wheat, such as Pm7, Pm8, Pm17, Pm20, and Pm56 derived from S. cereale [33–35]; Pm21, Pm55, Pm62, and Pm67 are from D. villosum [10,36–38]. Pm97033 [39] and 92R178 [40] are wheat lines carrying strong powdery mildew (PM) resistance genes derived from different accessions of D. villosum. The alien chromosome arms of 6V#4S and 6V#2S were translocated to different chromosomes of wheat, replacing 6DS [41] and 6AS [42–44], respectively. Genes from two homologous chromosome arms of this wild species are endowed with broad-spectrum resistance to PM, making them difficult to distinguish from each other and considered to be the same for a long time, because they were in different complex backgrounds. The powdery mildew resistance gene in the 6V#2S translocation line is Pm21 [10]. However, there is a different powdery mildew resistance gene in the 6V#4S translocation line. Before that, because they were all in the 6V translocation line, they were mistaken for the same disease resistance gene. In recent years, the Pm gene on 6V#4S has been confirmed and named as PMV [45,46]. Although there are many in-depth research studies on these two translocation lines, such as the development of markers [47,48], there are still no reports on the agronomic traits and Pm-resistant spectrum of these two translocation lines. In order to compare the genetic effects of the two translocated chromosomes on the PM-resistant spectrums, and to find out if there are any agronomic traits such as plant height, spike length, spikelet number, tillering ability, grain size, etc., linked with the alien chromosome arms, the two different translocation chromosomes must be studied under a similar wheat background. The wheat 660K SNP array is a useful genomic research tool. It has 660,000 probes that are almost evenly distributed across the genome [49]. Ninety-one percent of the probes have reliable physical positions. It can be used for high-throughput analysis with relatively low price. The probes have been widely used to screen bulked extreme phenotype DNA pools, develop Kompetitive Allele-Specific PCR (KASP) markers and simple sequence repeat (SSR) markers [50,51], and identify quantitative trait loci (QTL) [52–57], agronomic traits such as resistance to low nitrogen and genetic architecture of grain yield [58,59], and disease resistance such as wheat take-all, stripe rust [60–62] in common wheat. Recently, these probes have also been applied to determine the relationships of homoeologous chromosomes between wheat and its wild relatives [60,63]. In this study, two 6V#2S.6AL and 6V#4S.6DL translocation lines, A303 and B303, respectively bred using Pm97033 and 92R178 as parents backcrossed with wheat variety Wan7107, were scanned using the wheat 660k SNP chip. A total of 89,167 poly high- resolution probes were selected to analyze genomes of the three lines and the SNPs between the translocation lines and the recurrent parent Wan7107. In addition, the breakpoints of wheat chromosomes involved in translocations were determined. The distributions of SNPs on each chromosome between the translocations and Wan7107 were further investigated. Finally, under the same wheat background, the responses of two translocation lines to different strains of Bgt and their important agronomic traits such as plant height, spike length, spikelet number, tillering ability, grain size etc. were investigated and compared. The results of this study are valuable for understanding of genomic variation after continuous backcross, making effective use of the alien chromosome translocation lines, important genes, or to formulate breeding strategies. Agronomy 2021, 11, x FOR PEER REVIEW 3 of 19

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2. Materials and Methods 2.1. Plant Materials 2. Materials and Methods 2.1.Wan7107 Plant Materials is a mutation line derived from a naturally mutated ear of Funo, an Italian wheatWan7107 variety introduced is a mutation into line China derived in 1956 from by a naturally the Nanyang mutated Institute ear of Funo,of Agricultural an Ital- Sci- encesian wheat in Henan variety Province introduced [64]. into Funo China was in deri 1956ved by thefrom Nanyang the hybrid Institute of two of Agricul-Italian wheat varietiestural Sciences Duecentodievi in Henan Provinceand Damiano [64]. Funo and was preser derivedved at from the the Institute hybrid of twoPlant Italian Protection, Chinesewheat varieties Academy Duecentodievi of Agricultural and Damiano Sciences and (IPP, preserved CAAS). at the The Institute pedigree of Plant of Pro- A303 is Pm97033/Wan7107tection, Chinese Academy × 3 BC2 ofF4 Agricultural, in which Pm97033 Sciences is (IPP, a 6V#4S.6DL CAAS). The translocation pedigree of A303 line, is its ped- Pm97033/Wan7107 × 3 BC F , in which Pm97033 is a 6V#4S.6DL translocation line, its igree is TH3 (an amphydiploid2 4 of T. durum and D. villosum)/Wan7107 × 4 F5 [29]. The ped- pedigree is TH3 (an amphydiploid of T. durum and D. villosum)/Wan7107 × 4 F5 [29]. The igree of B303 is 92R178/Wan7107 × 9 BC8F4, bred, and preserved by professor Chen Xiao, pedigree of B303 is 92R178/Wan7107 × 9 BC8F4, bred, and preserved by professor Chen inXiao, which in which92R178 92R178 is a 6V#2S.6AL is a 6V#2S.6AL translocation translocation line, line, bred bred by NanjingNanjing Agricultural Agricultural Uni- Univer- sityversity and andprovided provided by by Professor Professor Chen Peidu.Peidu. The The relationships relationships among among Funo, Funo, Wan7107, Wan7107, A303,A303, and and B303 B303 are shownshown in in Figure Figure1. 1.

Figure 1. Genealogical diagram of the materials in this study. Figure 1. Genealogical diagram of the materials in this study.

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2.2. Genotyping Genomic DNA was isolated from 20 µg of leaves using a Plant Genomic DNA Kit (4992201/4992202) from Tiangen (Beijing, China) according to the manufacturer’s protocol. The DNA quality was detected using 1% Invitrogen E-Gel EX precast agarose gels (Thermo Fisher Scientific, Waltham, MA, USA). Qualified DNA samples were used for 660k SNP detection by Capitalbio Technology Corporation. Genotypic data were extracted and processed using the Axiom Analysis Suite 3.1.51. SNP with the highest typing reliability were selected, only polyhigh resolution probes (SNPs passed all thresholds) were used for analysis, and probes without corresponding physical locations were eliminated; only those with clear physical locations were retained. Finally, a total of 89,167 probes located on 21 chromosomes of three subgenomes of wheat were used to genotype the three materials. The average probe density on each chromosome is indicated in Figure2. Two types of poly high-resolution probes were used for SNP analysis of A303, B303, and Wan7107. The first type includes all genotypes of the recurrent parent Wan7107, with 89,167 probes (Table1). The second type eliminates all heterozygous genotypes of Wan7107 from the first class, with a total of 87,296 probes (Table2).

Table 1. Genotyping ratio on each chromosome in Wan7107.

NA AA AC AG AT CC CG GG TC TG TT Wan7107 (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) 1A 0.66 19.21 0.14 0.39 0.04 30.44 0.06 29.33 0.53 0.07 19.13 2A 0.46 17.99 0.09 0.41 0.00 32.08 0.00 30.61 0.41 0.03 17.91 3A 0.57 18.78 0.16 0.55 0.02 30.38 0.08 30.12 0.35 0.12 18.85 4A 0.44 19.35 0.07 0.39 0.00 29.87 0.13 30.71 0.33 0.05 18.68 5A 0.53 18.07 0.11 0.32 0.04 31.73 0.11 30.11 0.46 0.15 18.34 6A 0.59 18.74 0.30 1.14 0.00 29.67 0.34 27.89 1.19 0.25 19.88 7A 0.59 22.78 0.10 0.64 0.04 26.81 0.13 26.16 0.51 0.08 22.16 1B 0.39 19.23 0.12 0.73 0.05 29.76 0.02 29.59 0.71 0.05 19.35 2B 1.11 18.55 0.24 1.02 0.02 30.18 0.16 29.37 0.87 0.34 18.15 3B 0.68 19.49 0.12 0.51 0.02 29.91 0.17 28.39 0.50 0.07 20.12 4B 0.43 15.74 0.09 0.37 0.03 32.37 0.09 34.30 0.22 0.09 16.26 5B 0.49 19.43 0.10 0.49 0.00 29.75 0.04 29.45 0.48 0.16 19.61 6B 0.49 15.98 0.19 0.50 0.02 33.42 0.06 32.87 0.44 0.13 15.90 7B 0.48 17.97 0.13 0.67 0.08 31.36 0.19 30.42 0.56 0.13 18.00 1D 0.50 17.06 0.17 0.99 0.06 32.36 0.28 30.49 0.88 0.33 16.90 2D 0.39 14.11 0.26 1.74 0.00 34.99 0.58 34.02 0.97 0.32 12.63 3D 0.81 16.48 0.16 0.57 0.00 31.66 0.32 31.18 0.89 0.32 17.61 4D 0.71 20.93 0.00 0.63 0.08 27.25 0.16 30.41 0.71 0.08 19.04 5D 0.41 15.85 0.06 0.53 0.06 31.47 0.00 33.29 0.41 0.23 17.67 6D 0.00 16.69 0.38 0.99 0.00 31.87 0.30 33.08 1.21 0.30 15.17 7D 0.29 17.76 0.10 0.34 0.00 31.95 0.00 29.95 0.54 0.15 18.93 Average 0.53 18.10 0.15 0.66 0.03 30.92 0.15 30.56 0.63 0.17 18.11

Table 2. Number, density, and proportion of SNP probes on each chromosome in homozygous Wan7107.

Physical Number A303 vs. Wan7107(SNP) B303 vs. Wan7107(SNP) Density Density Proportion Density Proportion Chromosome Range of (kb) (Mb) Probes Number (kb) (%) Number (kb) (%) 1A 593.07 6949 85.35 34 17,443.24 0.49 91 6517.25 1.31 2A 780.26 6327 123.32 49 15,923.67 0.77 72 10,836.94 1.14 3A 750.55 4871 154.09 30 25,018.33 0.62 59 12,721.19 1.21 4A 743.86 6151 120.93 31 23,995.48 0.5 50 14,877.20 0.81 5A 709.42 5234 135.54 36 19,706.11 0.69 93 7628.17 1.78 6A 616.46 2359 261.32 25 24,658.40 1.06 648 951.33 27.47 7A 736.44 7072 104.13 40 18,411.00 0.57 107 6882.62 1.51 1B 688.58 4113 167.42 24 28,690.83 0.58 46 14,969.13 1.12 2B 801.18 5516 145.25 43 18,632.09 0.78 77 10,404.94 1.4 3B 830.14 9382 88.48 40 20,753.50 0.43 92 9023.26 0.98 4B 673.24 3222 208.95 6 112,206.67 0.19 30 22,441.33 0.93 Agronomy 2021, 11, x FOR PEER REVIEW 5 of 20

3A 750.55 4871 154.09 30 25,018.33 0.62 59 12,721.19 1.21 4A 743.86 6151 120.93 31 23,995.48 0.5 50 14,877.20 0.81 5A 709.42 5234 135.54 36 19,706.11 0.69 93 7628.17 1.78 6A 616.46 2359 261.32 25 24,658.40 1.06 648 951.33 27.47 7A 736.44 7072 104.13 40 18,411.00 0.57 107 6882.62 1.51

1B 688.58Agronomy 20214113, 11 , 399 167.42 24 28,690.83 0.58 46 14,969.13 1.12 5 of 19 2B 801.18 5516 145.25 43 18,632.09 0.78 77 10,404.94 1.4 3B 830.14 9382 88.48 40 20,753.50 0.43 92 9023.26 0.98

4B 673.24 3222 208.95 6 112,206.67Table 2. Cont0.19. 30 22,441.33 0.93 5B 712.96 6911 103.16 33 21,604.85 0.48 81 8801.98 1.17 Physical Number A303 vs. Wan7107(SNP) B303 vs. Wan7107(SNP) 6B 720.39 6382 112.88 35 Density20,582.57 0.55Density Proportion81 8893.70 Density1.27 Proportion Chromosome Range of (kb) Number Number 7B 750.54 3734 (Mb)201 Probes12 62,545.00 0.32(kb) 35(%) 21,444.00 (kb)0.94 (%) 5B 712.96 6911 103.16 33 21,604.85 0.48 81 8801.98 1.17 1D 495.15 6B1817 720.39272.51 63826 112.8882,525.00 35 0.33 20,582.57 17 0.55 29,126.47 81 8893.700.94 1.27 2D 651.43 7B1552 750.54419.74 37347 20193,061.43 12 0.45 62,545.00 15 0.32 43,428.67 35 21,444.000.97 0.94 1D 495.15 1817 272.51 6 82,525.00 0.33 17 29,126.47 0.94 3D 614.95 2D1238 651.43496.73 15522 419.74307,475.00 7 0.16 93,061.43 13 0.45 47,303.85 15 43,428.671.05 0.97 3D 614.95 1238 496.73 2 307,475.00 0.16 13 47,303.85 1.05 4D 508.64 4D1266 508.64401.77 126614 401.7736,331.43 14 1.11 36,331.43 15 1.11 33,909.33 15 33,909.331.18 1.18 5D 565.47 1703 332.04 11 51,406.36 0.65 21 26,927.14 1.23 5D 565.47 6D1703 473.51332.04 131811 359.2651,406.36 147 0.65 3221.16 21 11.15 26,927.14 28 16,911.071.23 2.12 6D 473.51 7D1318 638.31359.26 2050147 311.373,221.16 911.15 70,923.33 28 0.44 16,911.07 15 42,554.002.12 0.73 7D 638.31 2050 311.37 9 70,923.33 0.44 15 42,554.00 0.73

Figure 2.FigureDistribution 2. Distribution of 89,167 of probes 89,167 used probes for the used genotyping. for the genotyping. The abscissa The shows abscissa the chromosome, shows the the left ordinate shows thechromosome, number of probes the left on ordinate each chromosome, shows the and number the right of probes ordinate on shows each thechromosome, density of the and number the right of probes on ordinate shows the density of the number of probes on each chromosome. The orange bars each chromosome. The orange bars represent the number of probes, and the gray polyline shows the trend of density on represent the number of probes, and the gray polyline shows the trend of density on each each chromosome. chromosome. 2.3. Field Experimental Design and Investigation of Agronomic Characters 2.3. Field Experimental Design and Investigation of Agronomic Characters A303, B303, and Wan7107 were planted at the experimental station from March to A303, B303,June and inWan7107 2019 in ICS, were CAAS, planted Beijing, at China.the experimental Three randomized station block from repeats March were to designed. June in 2019 in ICS,Each CAAS, material Beijing, was planted China. in Three three randomized rows with 30 block seeds inrepeats each row were that designed. were sown evenly, Each material wasat planted a row spacing in three of 30 rows cm. with At maturity, 30 seeds ten in plants each perrow material that were were sown randomly evenly, collected to at a row spacinginvestigate of 30 cm. theAt plantmaturity, height, ten ear plants length, per ear material number, numberwere randomly of spikelets, collected number of grains to investigate theper plant ear, numberheight, ofear sterile length, spikelets, ear number, and thousand number grain of weight. spikelets, number of × grains per ear, numberThree of sterile BC2F3 spikelets,and BC2F4 andpopulations thousand derived grain from weight. a cross of Pm97033/Wan7107 3 were planted in a greenhouse from October 2018 to February 2019, at ICS, CAAS, in Beijing Three BC2F3 and BC2F4 populations derived from a cross of Pm97033/Wan7107 × 3 to evaluate the agronomic traits. In this experiment, the PM resistance in three populations were planted inwas a greenhouse still in segregation. from October Plants in 2018 each population to February were 2019, divided at ICS, into CAAS,two types: in resistant Beijing to evaluateand the susceptible. agronomic The resistanttraits. In type this is experiment, considered to the contain PM the resistance translocation in three chromosomes, populations waswhile still the in segregation. susceptible type Plants is not. in Both each types population of plants were were derived divided from into a two backcrossing types: resistant population, and susceptible. they have The similar resistant background type except is for considered the alien chromosome to contain arm, the so it can be compared for the agronomic traits associated with the alien chromosome arm. Each plant was investigated for plant height, ear length, ear number, number of spikelets, number of grains per ear, ear number, number of sterile spikelets, sterile ﬂoret number, and grain Agronomy 2021, 11, 399 6 of 19

weight during harvest. Comparisons of agronomic characters were conducted between the resistant and the susceptible plants.

2.4. Statistical Analysis IBM SPSS version 20.0.0 software was used to conduct t-tests. Graphs and tables were generated in R Studio (version 3.6.0, Boston, MA, USA) and Microsoft Excel (2019, Redmond, DC, USA).

2.5. Evaluation of PM Resistance of the Translocation Lines and Wheat Wan7107 2.5.1. Development of Conidia of Bgt on Plant Leaves To compare the PM resistance between the translocation lines and Wan7107, spores of Blumeria graminis f. sp. tritici (Bgt) were inoculated on leaves of three materials according to the method described by Li et al. [48]. The Coomassie brilliant blue staining method was used to stain the wheat leaf segments harvested at 25 h and 72 h after inoculation. First, the leaf segments were put into ﬁxative solution (ethanol: glacial acetic acid = 1:1) for 24 h. Then, the leaves were removed, washed with tap water, and put in decolorizing solution (lactic acid: glycerin: water = 1:1:1) for 48 h. The decolorizing solution was replaced every 24 h to make the leaf segments appear transparent and ensure complete decolorization. Then, leaves were dyed with 0.6% Coomassie Brilliant Blue R250 staining solution for 5 min and then rinsed with tap water. Finally, the leaf segments were stored in a preservation solution (glacial acetic acid: glycerol: water = 1:4:15). The development of conidia on the dyed wheat leaf segments was observed under an optical microscope. The experiment was repeated three times.

2.5.2. Reaction to Different Isolates of the Pathogen To know whether there are differences in the resistance between two translocations, the PM susceptible wheat variety “Chancellor”, which does not contain any PM resistance genes, was seeded in a 10 cm diameter ﬂowerpot to propagate the pathogen strains. Two tested translocation lines were seeded in a plastic box of 36 cm × 25 cm × 10 cm, and about 10 seeds were sown for each line with susceptible material “Funo” and resistant material “Nannong9918” as controls; Nannong9918 is a wheat variety carrying the 6V#2S.6AL translocation chromosome. Its pedigree is Yangmai158/92R137(6V#S.6AL)//Yangmai158, bred by Nanjing Agricultural University. In order to prevent wheat seedlings from being infected with other mixed pathogen isolates, transparent plastic bags covered with an iron wire frame were used to form a closed space, and they were cultured in a greenhouse about 20 ◦C for 8–10 days. When the wheat grew to the one-leaf stage, fresh spores of the pathogen were inoculated evenly on the wheat seedlings using the shaking method. In total, 24 different isolates of the pathogen were used. Each isolate of Bgt was inoculated into each tested line, and plants were cultivated in a greenhouse for 10–12 days to investigate the disease symptoms. The resistance level was graded according to Si Quanmin with “0–4” level [65]. “0” represents immune type, no disease spots seen by eyes; “0;”represents hypersensitive type, with white or yellow brown necrotic spots on the leaves, some with sparse short hyphae around the spots; Grade “1”: disease-resistant type, the hyphae on the surface of the lesion were thin with few conidia; Grade “2”: moderately resistant, with moderate hyphae and few conidia, lesions few or small, severity below 5%, and some with a chlorotic halo; Grade “3”: moderately susceptible, the hyphae on the surface of the lesion developed moderately to vigorously, with more spots and more conidia, but the lesions were not connected; Grade “4”: susceptible type, the hyphae on the surface of the disease spot were extensive, a large amount of spores was produced, and the lesions were mostly connected. Types 0–2 were non-pathogenic isolates and wheat varieties (or lines) were resistant; types 3–4 were virulent isolates, and wheat varieties (or lines) were susceptible. Agronomy 2021, 11, x FOR PEER REVIEW 7 of 19

were extensive, a large amount of spores was produced, and the lesions were mostly connected. Types 0–2 were non-pathogenic isolates and wheat varieties (or lines) were resistant; types 3–4 were virulent isolates, and wheat varieties (or lines) were susceptible.

3. Results 3.1. Characteristics of Base Composition in the Subgenome by Genotyping the Three Lines The genotyping results obtained using 89,167 probes showed that the homozygous Agronomy 2021, 11, 399 sites in the genome of Wan7107 occupied 97.69% of the total probes, among7 ofwhich 19 CC, GG, AA, and TT genotypes accounted for 30.92%, 30.56%, 18.10%, and 18.11%, respectively. The number of invalid probes on chromosome 2B was the largest in the genome (Table 1).3. Results The3.1. proportion Characteristics of of homozygous Base Composition and in the heteroz Subgenomeygous by Genotyping loci and the their Three distribution Lines in the genomes ofThe 6V#4S.6DL genotyping translocation results obtained line using A303 89,167 was probes very showed similar that to thethat homozygous of Wan7107, with 97.89% sitesof the in thehomozygous genome of Wan7107 loci (Table occupied S1). 97.69% However, of the total chromosome probes, among 6D which is an CC, exception: GG, its proportionAA, andof invalid TT genotypes probes accounted was the forhighes 30.92%,t of 30.56%, all chromosomes; 18.10%, and 18.11%, homozygous respectively. genotypes The number of invalid probes on chromosome 2B was the largest in the genome (Table1). decreased; theThe proportion of of homozygous its AA genotype and heterozygous was the lowest, loci and and their its distribution TT genotype in the was the second genomeslowest of 6V#4S.6DLall chromosomes. translocation On line the A303 contrary, was very the similar proportion to that of Wan7107,of its heterozygous with genotypes97.89% increased. of the homozygous The percentages loci (Table of S1). each However, of its chromosomeheterozygous 6D isgenotypes an exception: were its all the highestproportion among 21 of chromosomes. invalid probes was Chromosome the highest of all 2B chromosomes; still retained homozygous a higher proportion genotypes of in- decreased; the proportion of its AA genotype was the lowest, and its TT genotype was the valid probessecond such lowest as of Wan7107. all chromosomes. On the contrary, the proportion of its heterozygous Thegenotypes percentage increased. of homozygous The percentages and of heterozygous each of its heterozygous genotypes genotypes in the were6V#2S.6AL all the trans- locationhighest line B303 among and 21 its chromosomes. distribution Chromosome characteristics 2B still in retainedthe genome a higher were proportion also very of similar to that invalidof Wan7107. probes such The as average Wan7107. homozygous genotypes of AA, TT, CC, and GG were The percentage of homozygous and heterozygous genotypes in the 6V#2S.6AL translo- 17.85%, 17.92%, 30.77%, and 30.43%, respectively, and the total homozygous sites ac- cation line B303 and its distribution characteristics in the genome were also very similar to countedthat for of 96.97% Wan7107. of The all average genotypes. homozygous The si genotypesgnificant of AA,difference TT, CC, andcame GG from were 17.85%,chromosome 6A, and17.92%, the proportion 30.77%, and of 30.43%, its invalid respectively, probes and was the the total highest homozygous of all siteschromosomes. accounted for The pro- portions96.97% of its of four all genotypes.homozygous The signiﬁcantgenotypes difference were the came lowest from of chromosome all chromosomes, 6A, and the while the proportionproportion of its ofheterozygous its invalid probes genotypes was the highest had a of coincident all chromosomes. increase The proportions(Table S2). of Genotypes its four homozygous genotypes were the lowest of all chromosomes, while the proportion of AC, AG,its heterozygousTC, and TG, genotypes but not hadAT aand coincident CG in increase 6A, were (Table the S2). highest Genotypes of AC,all AG,chromosomes. TC, Figure and3 shows TG, but the not genotyping AT and CG in proportion 6A, were the of highest the 21 of allchromosomes chromosomes. Figurein Wan71073 shows and the two translocationthe genotyping lines proportion by 660k of thechip. 21 chromosomesAlthough the in overall Wan7107 base and the composition two translocation characteristics of thelines three by 660k lines chip. are Although very similar, the overall there base are composition significan characteristicst differences of among the three the lines chromo- are very similar, there are signiﬁcant differences among the chromosomes in homologous somes in homologous group 6, especially with chromosomes 6D and 6A. group 6, especially with chromosomes 6D and 6A.

Figure 3. PercentagesFigure of genotypes 3. Percentages on the 21 of chromosomes genotypes in on A303, the Wan7107, 21 chromo andsomes B303. Chromosome in A303, Wan7107, 6A is shown and in redB303. Chromo- and chromosome 6Dsome is shown 6A is in shown black. in red and chromosome 6D is shown in black.

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3.2. Snps between Wheat Wan7107 and Translocation Lines 3.2. SnpsTo betweencompare Wheat the Wan7107differences and between Translocation the Linestwo translocation lines A303 and B303 with Wan7107,To compare an SNP the analysis differences was between carried the out. two Th translocatione numbers, linesdensities, A303 andand B303 proportion with of the Wan7107,SNP probes an SNP to the analysis total wasnumber carried of out.the probes The numbers, per chromosome densities, and are proportion listed in ofTable the S3. The SNPnumber probes of tothe the SNPs total numberin the D of genome the probes is perthe chromosome lowest, and are the listed probe in Tabledensity S3. Theis the rarest number(Figure of2). the However, SNPs in the Dtotal genome number is the of lowest,probes andin each the probechromosome densityis of the the rarest D genome is (Figurethe lowest,2). However, so the theproportion total number of SNPs of probes in inthe each D genome chromosome is similar of the D to genome that in is the the A and B lowest, so the proportion of SNPs in the D genome is similar to that in the A and B genomes. genomes. When comparing A303 to Wan7107, chromosome 6D is an exception because it When comparing A303 to Wan7107, chromosome 6D is an exception because it contains manycontains more many SNPs more than otherSNPs chromosomes: than other chromo the numbersomes: of the SNPnumber probes of accountsthe SNP for probes ac- 13.2%counts of for all probes13.2% onof thisall probes chromosome, on this which chromosome, is the highest which of all is chromosomes the highest of in theall chromo- genome.somes in In the contrast, genome. when In comparing contrast, B303 when to Wan7107,comparing the B303 SNPs ofto chromosomeWan7107, the 6A SNPs made of chro- upmosome 30.35% 6A of the made total up probes, 30.35% which of the was total the highest probes, of which all chromosomes was the highest in the genome of all chromo- (Figuresomes4 in). the genome (Figure 4).

FigureFigure 4. 4Schematic. Schematic diagram diagram of the of percentagethe percentage of SNPs of between SNPs between two translocation two translocation lines and Wan7107 lines and onWan7107 each chromosome. on each chromosome.

3.3.3.3. Distribution Distribution of of SNPs SNPs on Chromosomeson Chromosom betweenes between Wan7107 Wan7107 and Translocation and Translocation Lines Lines The distribution of SNPs between A303 and Wan7107 in each chromosome arm The distribution of SNPs between A303 and Wan7107 in each chromosome arm showed that the average SNP distance on chromosome 6D was about 2715.639 kb, in whichshowed 168 that of 174 the polymorphic average SNP probes distance were denseon chromosome in the physical 6D intervalwas about of 53.099 2715.639 kb kb, in towhich 211.975 168 Mb, of 174 accounting polymorphic for 96.55% probes of the were total dense SNPs. in The the SNP physical density interval in this regionof 53.099 kb to reaches211.975 1261.438 Mb, accounting kb per probe for (Figure 96.55%5). Thereof the are total 716 SNPsSNPs. between The SNP B303 density and Wan7107 in this region onreaches chromosome 1261.438 6A, kb with per an probe average (Figure distance 5). ofThere 841.169 are kb. 716 However, SNPs between 682 SNPs B303 are denseand Wan7107 inon the chromosome physical region 6A, of with 632.649 an average kb to 282.152 distance Mb, accounting of 841.169 for kb. 95.25% However, of the 682 total SNPs SNPs, are dense whichin the makesphysical the region SNP density of 632.649 of this kb interval to 282.152 reach Mb, 412.786 accounting kb per probe. for 95.25% The remaining of the total SNPs, 34which SNPs makes were distributed the SNP indensity the long of arm this in interval the range reach of 286.037–617.097 412.786 kb per Mb, probe. among The which remaining there are 13 probes in the region of 431.834–436.981 Mb, with 12 SNPs, which increases the 34 SNPs were distributed in the long arm in the range of 286.037–617.097 Mb, among SNP density to 428.904 kb per probe (Figure6). which there are 13 probes in the region of 431.834–436.981 Mb, with 12 SNPs, which increases the SNP density to 428.904 kb per probe (Figure 6).

AgronomyAgronomy 2021, 202111, x, 11FOR, 399 PEER REVIEW 9 of 199 of 19 Agronomy 2021, 11, x FOR PEER REVIEW 9 of 19

FigureFigureFigure 5. Distribution 5. 5. Distribution Distribution of SNPs of of SNPs on SNPs each on on chromosome each each ch chromosomeromosome between between A303 between and A303 Wan7107.A303 and and Wan7107. Wan7107.

Figure 6. Distribution of SNPs on each chromosome between B303 and Wan7107. FigureFigure 6. 6. Distribution Distribution of of SNPs SNPs on on each each ch chromosomeromosome between between B303 B303 and and Wan7107. Wan7107.

InIn addition addition to to the the key key chromosomes chromosomes and and th theireir homologous homologous chromosomes chromosomes in in group group 6, 6, aa SNP SNP dense dense region region (371.362 (371.362 kb kb to to 26.564 26.564 Mb) Mb) was was observed observed at at the the telomere telomere region region of of 2BS 2BS inin both both translocation translocation lines. lines. There There are are 135 135 SNPs SNPs in in this this region region of of A303, A303, with with an an average average densitydensity of of 183.355 183.355 kb, kb, which which is is higher higher than than the the average average density density value value of of chromosome chromosome 2B 2B (5,722.712(5,722.712 kb). kb). The The telomere telomere region region of of the the 2BS 2BS chromosome chromosome of of the the 6V#2S.6AL 6V#2S.6AL translocation translocation lineline B303 B303 contained contained 147 147 SNPs, SNPs, with with an an average average density density of of 178.177 178.177 kb, kb, which which is is much much higher higher

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In addition to the key chromosomes and their homologous chromosomes in group 6, a SNP dense region (371.362 kb to 26.564 Mb) was observed at the telomere region of 2BS in both translocation lines. There are 135 SNPs in this region of A303, with an average density of 183.355 kb, which is higher than the average density value of chromosome 2B (5722.712 kb). The telomere region of the 2BS chromosome of the 6V#2S.6AL translocation line B303 contained 147 SNPs, with an average density of 178.177 kb, which is much higher than the average density value of chromosome 2B (3576.695 kb). In addition, there are 14 consecutive SNPs on chromosome 2A from 600.423 to 605.534 Mb when comparing A303 to Wan7107; the average density value of SNPs in this region is 365.107 kb. In the comparison of B303 and Wan7107, there are 40 SNPs in the region of 663.328 to 669.909 Mb on chromosome 5A; the SNP density in this region is 164.631 kb. There are 23 SNPs in the region of 20.448 to 26.241 Mb on chromosome 7A; the density of this region is 251.870 kb. Overall, the SNP densities of these regions are significantly higher than the average densities of the chromosomes. In total, there are 1775 polymorphic loci between A303 and Wan7107, which occupy 1.99% of the total 89,167 probes. There are 2902 polymorphic loci between B303 and Wan7107, which account for 3.25% of 89,167 of the total probes. All of them are higher than expected. Moreover, the SNPs between B303 and Wan7107 are higher than those between A303 and Wan7107, which is not consistent with the theoretical expectation. Considering that anther culture was applied in the breeding process of Pm97033, this should be a key step to improve homozygosity, leading to decreased SNPs between A303 and Wan7107 compared to those between B303 and Wan7107. Therefore, it is reasonable to speculate that the higher heterozygous sites of Wan7107 itself are the main reason for the actual SNP value between the two translocation lines and Wan7107 being higher than expected. In order to confirm this speculation, we filtered out all the heterozygous genotypes of Wan7107 and used only the homozygous sites for SNP analysis. The results showed that the probes of SNP between A303 and Wan7107, and that between B303 and Wan7107 account for 1.06% and 2.44% of the total probes, respectively (Table2). When the SNPs of chromosome 6D in A303 and chromosome 6A in B303 were excluded, the average SNP probes of other chromosomes accounted for 0.56% and 1.19% of the total probes, respectively, which is very close to the expected value. It means that A303 and Wan7107 had 99.44% identical genotypes, except for the 6D chromosome; B303 and Wan 7107 had 98.81% identical genotypes, except for the 6A chromosome.

3.4. Differences in Powdery Mildew Resistance between Two Translocation Lines Translocation lines A303 and B303 were evaluated for reaction to PM pathogen compare to Wan7107. No secondary hyphae were observed in the primary germ tube and appressorium at 25 h after inoculation (Figure7), which indicated that the development of conidia on leaves of the translocation lines was largely limited, and it failed to infection in the translocation lines. However, it developed very fast and produced secondary hyphae at 25 h and many conidiophores at 72 h in the check line Wan7107 (Figure7). Agronomy 2021, 11, 399 11 of 19 Agronomy 2021, 11, x FOR PEER REVIEW 11 of 19

FigureFigure 7. 7.Developmental Developmental states states of theof the conidiospores conidiospores 25 h 25 and h 72and h after72 h inoculationafter inoculation on leaves on ofleaves of thethe translocationtranslocation lines. lines. (a )(a Spores) Spores 25 h25 after h after inoculation inoculation on leaves on leav of thees of translocation the translocation lines A303. lines A303. ((bb)) SporesSpores 72 72 h h after after inoculation inoculation on on leaves leav ofes theof the translocation translocation lines lines A303. A303. (c) Spores (c) Spores 25 h after25 h after in- inoculationoculation on leavesleaves of of the the translocation translocation lines lines B303. B303. (d) Spores (d) Spores 72 h after 72 inoculationh after inoculation on leaves ofon the leaves of translocationthe translocation lines B303. lines ( eB303.) Spores (e) 25Spores h after 25 inoculation h after inoculation on leaves of on Wan7107. leaves (off) SporesWan7107. 72 h ( afterf) Spores 72 h inoculationafter inoculation on leaves on ofleaves Wan7107. of Wan7107. White arrows White point arro tows spores, point yellow to spores, arrows yellow point arrows to primary point to pri- germinalmary germinal tubes, red tubes, arrows red point arrows to appressorium, point to appressorium, and green arrows and pointgreen to arrows secondary point hyphae. to secondary hyphae. To compare differences in disease resistance between the translocation lines, 24 strains of theTo pathogen compare were differences separately inoculatedin disease on resistance seedlings ofbetween the translocation the translocation lines. The lines, 24 results (Table3) showed that both translocation lines were immune (Grade “0”) or hyper- strains of the pathogen were separately inoculated on seedlings of the translocation lines. sensitive (Grade “0;”) to most of the strains except E15 and E20, to which both translocation linesThe showedresults different(Table 3) grades showed of resistance, that both i.e., translocation A303 reaction tolines E15 were was “0;“and immune B303 (Grade was “0”) or “2hypersensitive + 0;”, the reaction (Grade of A303 “0;”) to to E20 mo wasst of grade the “1“strains and except B303 was E15 grade”0;”. and E20, At to thewhich same both trans- time,location the susceptiblelines showed control different variety grades “Funo” of showed resistance, severe i.e., disease A303 symptoms reaction (Gradeto E15 “3”was “0;“and orB303 Grade was “4”), “2 + while 0;”, resistancethe reaction control of A303 variety to “Nannong9918”E20 was grade with“1“ and the Pm21 B303resistance was grade”0;”. At the same time, the susceptible control variety “Funo” showed severe disease symptoms (Grade “3” or Grade “4”), while resistance control variety “Nannong9918” with the Pm21 resistance gene under a different background showed immunity or hypersensitivity to all strains (Table 3).

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gene under a different background showed immunity or hypersensitivity to all strains (Table3).

Table 3. Reactions of translocation lines to 24 different individual Bgt strains.

Isolate of Bgt Funo A303 B303 Nannong9918 E01 4 0; 0; 0; E05 4 0; 0 0; E06 4 0; 0 0; E07 4 0; 0 0 E09 4 0 0; 0; E11 4 0; 0; 0; E13 4 0 0; 0; E15 3 0; 2+0; 0; E16 4 0 0 0; E17 4 0; 0 0; E18 4 0; 0; 0 E20 4 1 0; 0; E21 4 0 0; 0; E23-(1) 4 0 0; 0; E23-(2) 4 0 0; 0; E26 3 0; 0; 0 E30-(1) 4 0 0 0 E30-(2) 4 0; 0; 0; E31 4 0; 0 0; E32 4 0; 0; 0; E49 4 0; 0; 0 E50 3 0 0 0; E60 4 0 0 0; E69 4 0; 0 0;

3.5. Effects of Different Chromosome Translocations on Agronomic Traits In order to understand the effects of different exogenous chromosome arm translocations on agronomic traits under the same background, the main agronomic traits of A303, B303, and Wan7107 were investigated and compared. As shown in Table4, when 6AS or 6DS were substituted by the alien chromosome arm, it resulted in significantly increased plant height and grain weight in both translocation lines. Compared with Wan7107, the thousand-grain weights of the two lines of B303 were 5.29 and 6.78 higher, accounting for 15.66% and 20.07%, respectively (Figure8), while A303 increased by 17.44%. Plant height also increased significantly, and the two lines of B303 increased by 5.47% and 4.93%, while A303 had an increase of 9.71%. The ear length in A303 increased but the spikelet number and grain number per spike did not change, and its number of ears decreased slightly. Compared with the control, the number of spikelets in B303 was significantly lower, suggesting that 6V#2S replacement of 6AS might have a negative effect on spikelet number.

Table 4. Comparison of main agronomic traits between translocation lines and their recurrent parent Wan7107.

Materials Individuals PH(cm) EL(cm) SN GNPS EN SFN TGW(g) A303 30 78.55 ± 3.52 7.75 ± 0.96 19.63 ± 1.03 54.87 ± 6.61 4.33 ± 1.58 0.17 ± 0.38 39.67 ± 1.07 p value ** ** ns ns ns ns ** B303-1 30 75.52 ± 3.86 7.45 ± 0.86 19.20 ± 1.63 50.67 ± 7.09 4.63 ± 1.43 0.63 ± 0.76 39.07 ± 0.40 p value ** ns * * ns * ** B303-2 30 75.13 ± 4.67 7.11 ± 0.91 18.67 ± 1.44 52.90 ± 7.54 4.80 ± 1.39 0.43 ±0.62 40.56 ± 0.16 p value ** ns ** ns ns ns ** Wan7107 30 71.60 ± 2.85 7.15 ± 0.66 20.10 ± 1.52 55.67 ± 6.48 4.63 ± 1.49 0.23 ± 0.43 33.78 ± 0.83 Asterisks indicate significance determined by t-test for each population * p < 0.05, ** p < 0.01. ns: no significant difference. Abbreviations: PH: plant height; EL: ear length; SN: spikelet number; GNPS: grain number per spike; EN: ear number; SFN: sterile floret number; TGW: thousand grain weight. Agronomy 2021, 11, x FOR PEER REVIEW 13 of 19

Wan7107 30 71.60 ± 2.85 7.15 ± 0.66 20.10 ± 1.52 55.67 ± 6.48 4.63 ± 1.49 0.23 ± 0.43 33.78 ± 0.83 Asterisks indicate significance determined by t-test for each population *p < 0.05, ** p < 0.01. ns: no significant difference. Abbreviations:Agronomy 2021, 11, 399PH: plant height; EL: ear length; SN: spikelet number; GNPS: grain number per13 spike; of 19 EN: ear number; SFN: sterile floret number; TGW: thousand grain weight.

FigureFigure 8. Comparison8. Comparison of grain of shape grain between shape two between translocation two lines translocation and Wan7107. lines and Wan7107. To evaluate the effects of the translocations on agronomic traits under PM, the main agronomicTo evaluate traits the of three effects BC2F of3 and the BC translocatio2F4 populationsns derivedon agronomic from a cross traits of under PM, the main × Pm97033/Wan7107agronomic traits3 plantedof three in a greenhouseBC2F3 and were BC investigated.2F4 populations Plant height, derived spike from a cross of length, grain number per spike, floret number, and thousand grain weight of resistant plantsPm97033/Wan7107 were higher than those × 3 ofplanted susceptible in plants, a greenhouse while the numbers were of investigated. spikelets and Plant height, spike sterilelength, floret grain number number in the resistant per spike, plants werefloret reduced. number However,, and some thousand traits such grain as weight of resistant plantplants height were and higher spike length than in twothose lines of of susceptibl BC2F4 did note showplants, significant while differences the numbers of spikelets and between the resistant (R) and susceptible (S) plants. Increased sterile floret number in the disease-susceptiblesterile floret number plants indicated in the thatresistant the seed plants setting rate we wasre reduced. largely affected However, by the some traits such as diseaseplant (Tableheight5). Thisand result spike is basically length consistent in two withlines those of ofBC the2F field4 did investigation. not show significant differences between the resistant (R) and susceptible (S) plants. Increased sterile floret number in the Table 5. Comparison of main agronomic traits between resistant and susceptible plants of three BC2F3 and BC2F4 populations derived from a cross ofdisease-susceptible Pm97033/Wan7107 × 3 planted plants in a greenhouse. indicated that the seed setting rate was largely affected by the

Pm97033/ disease (Table 5). This result is basically consistent with those PMof the field investigation. Individuals PH EL SN GNPS FN EN SFN TGW Wan7107 Reaction ± ± ± ± ± ± ± ± BC F 56 82.98 6.25 6.08 0.51 21.19 1.57 33.32 8.22 43.30 5.79 1.96 0.87 3.91 1.52 44.82 0.18 R 2 3 34 78.41 ± 6.15 5.75 ± 0.64 22.00 ± 1.41 26.82 ± 7.25 40.47 ± 6.45 2.26 ± 0.86 6.12 ± 1.71 40.48 ± 0.16 S p value **Table ** 5. Comparison * of **main agronomic * traits ns between ** resistant ** and susceptible plants of three 22 82.50 ± 5.34 6.41 ± 0.52 21.05 ± 1.13 34.9 ± 5.84 47.41 ± 6.58 2.23 ± 1.15 3.5 ± 1.33 44.03 ± 0.03 R BC2F4-1 23 81.83 ± 3.02BC2 6.22F3 ±and0.44 BC 21.952F±4 1.11populations 29.71 ± 6.08 derived 43.52 ± 5.52 from 2.48 ± a0.73 cross 4.86 of± 1.28 Pm97033/Wan7107 40.16 ± 0.20 S × 3 planted in a green- p value ns ns * * * ns ** ** 22 81.35 ± 6.20house. 6.49 ± 0.61 20.23 ± 1.34 34.86 ± 9.93 45.27 ± 6.36 1.90 ± 1.15 3.77 ± 1.87 43.27 ± 0.73 R BC2F4-2 25 78.64 ± 5.90 6.26 ± 0.47 22.40 ± 1.00 31.62 ± 6.10 46.20 ± 3.91 2.00 ± 0.64 4.80 ± 1.52 36.66 ± 0.16 S p value ns ns ** ns ns ns * ** Pm97033/ AsterisksIndividuals indicate signiﬁcancePH determined byELt-test for each population,SN * p

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4.2. SNP Distribution between Translocation Lines and Recurrent Parent In the comparison of translocation lines and the recurrent parent, the dense regions of SNPs on key chromosomes were interrupted at the physical sites of about 211.975 Mb in 6D and 282.153 Mb in 6A, indicating that the breakpoints of chromosomes 6D and 6A were located at these locations. According to the report by Su et al. [66], the physical positions of the centromeres of 6A and 6D were at 283.3–288.7 Mb and 211.9–217.5 Mb, respectively. The breakpoint of 6DS in A303 is closely connected with the centromere, and that of 6AS in B303 is nearly 1.1 Mb from the centromere. Based on the results of the chip genotyping, the proportion of invalid probes on chromosome 2B was the highest in the genome of Wan7107. Although the SNPs on chromosome 2B accounted for a small proportion of the total number of probes (Table1), it is surprising that the SNPs between the translocation and near the distal part of 2BS were denser (Figures5 and6). The terminal region is a place where chromosome exchange occurs frequently, and after 7–9 generations of continuous backcrossing, there was still a high number of SNPs at the end of the chromosome, which indicates that in the natural population of Wan7107, the 2B chromosome maintains high heterozygosity. Therefore, despite multiple backcross generations, the high SNP number is still maintained, which is more prominent in the comparison between B303 and Wan7107 (Figure6). In the A303 translocation line, an anther culture was carried out during the breeding process, and the homozygosity of genotypes was greatly improved. In comparing A303 and Wan7107, the number of SNPs at the end of 2BS is slightly lower (Figure5). Another hypothesis is that 2BS has a large chromosome structure variation, or it has some homology with the 6VS chromosome, which needs further research. At the same time, we noticed that the existence of the alien in addition to the increased SNPs in the translocated chromosome itself also increased the SNPs in some other chromosomes. For example, the proportion of SNPs on 6D chromosome of A303 was as high as 11.15%, and the SNPs on 6A also increased to 1.06%. In addition to the expected SNP proportion of 6A chromosome increasing to 27.47%, the SNP proportions of 6D and 5A were signiﬁcantly higher than those of other chromosomes, which were 2.12% and 1.78%, respectively (Table2). This may be due to the homologous regions of foreign chromosomes on homoeologous or other wheat chromosomes. Although most of the gene sequences showed collinearity among species, some original homologous genes or sequences in different species might be located on non-homologous chromosomes [48].

4.3. 6VS Association with PM Resistance For a long time, PM resistance gene(s) on 6VS (including 6V#2S and 6V#4S) were considered the same due to the limitations of molecular markers. In fact, the genes derived from the homologous chromosome arms of the wild species confer broad-spectrum resistance to PM, making them difficult to distinguish from each other by the disease resistance phenotype, because they were in different complex backgrounds. Cao et al. cloned Stpk-V on chromosome 6V#2S and inferred it to be a key member at the Pm21 locus [67]. However, its homologous gene in 6V#4S had different sequences in the promoter region and intron [68]. He et al. [29] and Xing et al. [69] map-based cloned Pm21 from 6V#2S. Bie and He et al. [45] and Li et al. [70] identified PmV from 6V#4S and confirmed that this gene encodes a typical coiled-coil/nucleotide-binding site/leucine-rich repeat (CC-NBS-LRR) protein such as Pm21. In the present study, we found that their reactions to some of the stains of PM pathogen were also different. Here, “Nannong9918” is used as a PM resistance wheat control material. Similar to B303, it carries the pm21 resistance gene, but the resistance phenotypes are different from B303 due to their different genetic backgrounds. As far as we know, this is the first time to prove that the resistance of two 6VS to powdery mildew can be distinguished by race identification. Agronomy 2021, 11, 399 15 of 19

4.4. 6VS Association with Agronomic Characters In this study, we found that both translocation lines with the same wheat background not only had excellent PM resistance but also showed some desirable agronomic traits, such as increased grain weight. However, the plant height increased significantly, which is not conducive to lodging resistance. These results were consistent with the findings by Zhao et al. [45], who employed a recombinant inbred line (RIL) population constructed from the cross between T6V#2S·6AL translocation line ‘Yangmai180 and T6V#4S·6DL translocation line ‘Yangmai220 to evaluate the effects of the translocation chromosome on main agronomic traits. A303 and B303 also showed differences in some of these traits. For example, the ear length in A303 was significantly increased, but there was no large variation in B303; B303 had fewer spikelets than the control, while A303 did not have a significant change in number of spikelets. Under greenhouse conditions, PM disease occurrence was high when control measures were not taken. The plants with PM resistance genes had high seed setting rate and grain weight while susceptible plants were significantly affected by the disease. Many studies have shown that genes such as TaGW2-6A are related to grain development in 6AS [71–73]. The effect of replacing 6AS with 6VS seems to be beneficial to some yield traits but disadvantageous to others, so it needs to be further evaluated in terms of yield.

5. Conclusions Genotyping analysis of 660k SNP chip revealed that genotype characteristics of the two translocation lines of A303 and B303, and their background wheat parent Wan7107 were similar throughout the genomes except for the translocation chromosomes in A303 and B303. The translocation of 6V#4S and 6V#2S not only increased the proportion of invalid probes and heterozygous sites but also decreased the proportion of homozygous sites in the translocation chromosomes. The SNPs between the translocation lines and Wan7107 were signiﬁcantly increased and densely distributed in 6DS or 6AS and unexpectedly on the near telomere regions of 2BS. Moreover, A303 and Wan 7107 had 99.43% identical genotypes; B303 and Wan7107 had 98.78% identical genotypes. 6V#4S and 6V#2S have different reactions to some of the strains of Bgt. In addition, some important agronomic traits related to yield were affected by the alien chromosome arms.

Supplementary Materials: The following are available online at https://www.mdpi.com/2073-439 5/11/2/399/s1, Table S1: The proportion of homozygous and heterozygous loci and their distribution in the genomes of 6V#4S.6DL translocation line A303, Table S2: The percentage of homozygous and heterozygous genotypes in the 6V#2S.6AL translocation line B303, Table S3: Number and density of SNP probes and their proportion in the total probes on each chromosome. Author Contributions: Conceptualization, Z.L. and B.W.; formal analysis, B.W. and Z.L.; investigation, X.M. and Y.Z.; writing—original draft, B.W. and Z.L.; writing—review and editing, B.W., Z.L., and X.Y.; funding acquisition, Z.L., X.Y. and Y.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was financially supported by the National Key Research and Development Program of China (2016YFD0102001 and 2016YFD0102002), and the Agricultural Science and Tech- nology Innovation Program (ASTIP) of Chinese Academy of Agricultural Sciences (2060302-2-20). Acknowledgments: We would like to express our deep respects to Chen Xiao, and our present research is based on the materials she developed. We are grateful to Yiping Yan at ICS-CAAS for managing wheat plants and assisting with sampling. We also thank Chunkun Fan at Institute of Agriculture, Tibet Academy of Agricultural and Animal Husbandry Sciences, China, for helping to collect wheat samples post PM infection during his master’s degree at ICS-CAAS. And we want thank Huihui Li at ICS-CAAS for the advice of correlation analysis. Conflicts of Interest: The authors declare no conflict of interest. Agronomy 2021, 11, 399 16 of 19

Abbreviations

SNP Single Nucleotide Polymorphism QTL quantitative trait loci EN ear number RIL recombinant inbred line SFN sterile floret number TGW thousand grain weight EL ear length SNPS spikelet number per spike SN spike number FN floret number PH plant height GNPS grain number per spike KASP Kompetitive allele-specific PCR Bgt B. graminis f. sp. tritici PM powdery mildew SSR simple sequence repeat

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